Hereditary spherocytosis due to a novel frameshift mutation in AE1 cytoplasmic COOH terminal tail: band 3 Vesuvio.

Band 3 (anion exchanger 1, AE1) is the most abundant integral protein of the red blood cell membrane. It is composed of 911 amino acids encoded by the EPB3 gene. Several mutations in the EPB3 gene have been found associated to different diseases, eg, hereditary spherocytosis, Southeast Asian ovalocytosis, chorea-acanthocytosis, and familial distal renal tubular acidosis, thus demonstrating that the effects of EPB3 mutation are dependent on type and location.1,2 The function of the band 3 carboxyl terminus tail that protrudes on the cytoplasmic side of the erythrocyte membrane is unknown.3 We describe the first example of a disease-causing mutation located in the extreme 38 end of the AE1 coding sequence. Hereditary spherocytosis (HS) is a common hemolytic anemia in which the spheroidal, osmotically fragile erythrocytes are destroyed in the spleen. HS due to band 3 deficiency has an autosomal dominant mode of transmission and is characterized by mild to moderate anemia.1 Six children, belonging to unrelated Italian families from the Vesuvian area (Southern Italy), presented a homogeneous clinical picture showing moderate hemolytic anemia and splenomegaly. HS was diagnosed on the basis of clinical findings, familial history, and routine hematological investigations. The protein phenotype, performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), was typically that of band 3-deficient HS (band 3/spectrin: 221% 6 2% compared with that of the controls).4 In all patients, single/strand conformation polymorphism (SSCP) screening of band 3 gene showed an alteration in exon 20 (not shown).4 Direct sequencing of the abnormal fragments constantly showed a deletional frameshift mutation in codon 894 (ACC = AC; not shown). We designated this mutation with the area of origin as Vesuvio. Heteroduplex analysis confirmed this mutation in 5 additional patients in the 6 families. This analysis failed to find the mutation in 4 healthy members (Fig 1). In 2 brothers of family 3 we also detected a previously described silent polymorphism (codon 904, TAC = TAT)5 occurring in trans (Fig 1 and not shown). Analysis of the PstI polymorphic site of the EPB3 gene indicated that HS was invariably associated with a PstI (1) allele (not shown). Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) amplification with primers flanking the deletion (primer sense in exon 19 and primer antisense in exon 20) and with lower numbers of cycles produced heteroduplexes, suggesting that mRNA corresponding to allele Vesuvio was expressed (Fig 2). Urinary pH and acid excretion were in the normal range in all patients. Band 3 Vesuvio is the first mutation located in the cytoplasmic COOH terminal tail. The singularity of this mutation is the opening of the reading frame for an extra 133 codons (instead of 18), before the new stop codon at position 1027. The presence of the corresponding mRNA suggests that neither the transcription of the mutant gene nor the